Expression and purification of human respiratory syncytial virus recombinant fusion protein

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Human respiratory syncytial virus in children hospitalized for acute lower respiratory infection

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Diversity and Adaptation of Human Respiratory Syncytial Virus Genotypes Circulating in Two Distinct Communities: Public Hospital and Day Care Center

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Human respiratory syncytial virus detection in children admitted at a community hospital in Botucatu, SP, Brazil

O Vírus Respiratório Sincicial Humano (VRSH) é descrito como o mais importante patógeno viral causador de doenças respiratórias agudas das vias respiratórias inferiores em crianças. Neste estudo 84 amostras de crianças com idade abaixo dos dois anos apresentando sintomas de doença respiratória aguda, foram obtidas no período de setembro de 2000 a novembro de 2001. Analise por imunofluorescência indireta e transcrição reversa seguida de PCR, revelou que 18% (15/84) das amostras foram positivas, sendo que em 80% (12/15) dos casos a detecção de VRSH foi observada em crianças abaixo dos seis meses, e também que os subgrupos A e B co-circularam. Estes são os primeiros dados obtidos para a cidade de Botucatu, sendo que a sazonalidade mostrou-se evidente pela maior circulação desse vírus entre os meses de maio e julho

Clonagem e expressão da glicoproteína transmembrana do vírus linfotrópico de células T humanas em sistema procarioto

HTLV-1 is the virus that causes T cell lymphoma/leukemia in adults and a neurological disorder known as HTLV-associated myelopathy or tropical spastic paraparesis. One of the transmission means is through contaminated blood and its byproducts. Because of the risk of HTLV-associated infections, screening for HTLV was introduced for Brazilian blood donors in 1993. Most of the diagnostic kits used in the national blood banks are bought from foreign companies. Brazil does not have the technology to produce this material and there is a need to produce diagnostic systems with national technology. In this study, we show the expression of gp21/HTLV-1 in Escherichia coli and its reactivity towards monoclonal antibodies and the antibodies of infected patients. Expressing these proteins is the first step towards obtaining diagnostic kits with Brazilian biotechnology.

Positive selection results in frequent reversible amino acid replacements in the G protein gene of human respiratory syncytial virus

Human respiratory syncytial virus (HRSV) is the major cause of lower respiratory tract infections in children under 5 years of age and the elderly, causing annual disease outbreaks during the fall and winter. Multiple lineages of the HRSVA and HRSVB serotypes co-circulate within a single outbreak and display a strongly temporal pattern of genetic variation, with a replacement of dominant genotypes occurring during consecutive years. In the present study we utilized phylogenetic methods to detect and map sites subject to adaptive evolution in the G protein of HRSVA and HRSVB. A total of 29 and 23 amino acid sites were found to be putatively positively selected in HRSVA and HRSVB, respectively. Several of these sites defined genotypes and lineages within genotypes in both groups, and correlated well with epitopes previously described in group A. Remarkably, 18 of these positively selected tended to revert in time to a previous codon state, producing a flipflop phylogenetic pattern. Such frequent evolutionary reversals in HRSV are indicative of a combination of frequent positive selection, reflecting the changing immune status of the human population, and a limited repertoire of functionally viable amino acids at specific amino acid sites.

Molecular epidemiology of the SH (small hydrophobic) gene of human respiratory syncytial virus (HRSV), over 2 consecutive years

Human respiratory syncytial virus (HRSV) strains were isolated from nasopharyngeal aspirates collected from 965 children between 2004 and 2005, yielding 424 positive samples. We sequenced the small hydrophobic protein (SH) gene of 117 strains and compared them with other viruses identified worldwide. Phylogenetic analysis showed a low genetic variability among the isolates but allowed us to classify the viruses into different genotypes for both groups, HRSVA and HRSVB. It is also shown that the novel BA-like genotype was well segregated from the others, indicating that the mutations are not limited to the G gene. (C) 2011 Elsevier B.V. All rights reserved.

Diversity and Adaptation of Human Respiratory Syncytial Virus Genotypes Circulating in Two Distinct Communities: Public Hospital and Day Care Center

HRSV is one of the most important pathogens causing acute respiratory tract diseases as bronchiolitis and pneumonia among infants. HRSV was isolated from two distinct communities, a public day care center and a public hospital in Sao Jose do Rio Preto - SP, Brazil. We obtained partial sequences from G gene that were used on phylogenetic and selection pressure analysis. HRSV accounted for 29% of respiratory infections in hospitalized children and 7.7% in day care center children. On phylogenetic analysis of 60 HRSV strains, 48 (80%) clustered within or adjacent to the GA1 genotype; GA5, NA1, NA2, BA-IV and SAB1 were also observed. SJRP GA1 strains presented variations among deduced amino acids composition and lost the potential O-glycosilation site at amino acid position 295, nevertheless this resulted in an insertion of two potential O-glycosilation sites at positions 296 and 297. Furthermore, a potential O-glycosilation site insertion, at position 293, was only observed for hospital strains. Using SLAC and MEME methods, only amino acid 274 was identified to be under positive selection. This is the first report on HRSV circulation and genotypes classification derived from a day care center community in Brazil.

Human respiratory syncytial virus N, P and M protein interactions in HEK-293T cells

Characterization of Human Respiratory Syncytial Virus (HRSV) protein interactions with host cell components is crucial to devise antiviral strategies. Viral nucleoprotein, phosphoprotein and matrix protein genes were optimized for human codon usage and cloned into expression vectors. HEK-293T cells were transfected with these vectors, viral proteins were immunoprecipitated, and co-immunoprecipitated cellular proteins were identified through mass spectrometry. Cell proteins identified with higher confidence scores were probed in the immunoprecipitation using specific antibodies. The results indicate that nucleoprotein interacts with arginine methyl-transferase, methylosome protein and Hsp70. Phosphoprotein interacts with Hsp70 and tropomysin, and matrix with tropomysin and nucleophosmin. Additionally, we performed immunoprecipitation of these cellular proteins in cells infected with HRSV, followed by detection of co-immunoprecipitated viral proteins. The results indicate that these interactions also occur in the context of viral infection, and their potential contribution for a HRSV replication model is discussed.

Missed opportunities in the prevention of perinatal human immunodeficiency syndrome virus transmission

Mother to child transmission of human immunodeficiency virus (HIV) has decreased dramatically in the United States since the mid-1990s. Without antiretroviral therapy the risk of perinatal infection is as high as 25%; with treatment the risk drops to <1-2%. However, state surveillance data show a recent rise in the percentage of babies being born with HIV in Texas. No studies of perinatal HIV transmission in Texas have focused on the individual cases and identified what social/institutional barriers stood in the way of the index woman, her support system and her health providers in negotiating access to prenatal care and HIV treatment.^ The Texas Department of State Health Services identifies the babies born in Texas with HIV infection. This two year study will use mixed methods to identify barriers to the diagnosis and treatment of maternal HIV. In-depth interviews and chart reviews will be used to conduct the study. The abstracted medical record will give us demographic data and details of the timing of testing and treatment; interviews will provide information as to the individual and environmental factors that may have delayed testing and treatment. Little research has been done to assess the factors contributing to late prenatal HIV diagnosis and care in Texas and the interventions identified by mothers of affected babies that might overcome these obstacles.^ Conclusions from this study will guide the development of interventions to better educate the public, reduce structural barriers common to the underserved, and/or educate health care professionals. The study will also serve as a model for other states to undertake evaluation of their cases of perinatal infection. ^

A novel virus in swine is closely related to the human hepatitis E virus

A novel virus, designated swine hepatitis E virus (swine HEV), was identified in pigs. Swine HEV crossreacts with antibody to the human HEV capsid antigen. Swine HEV is a ubiquitous agent and the majority of swine ≥3 months of age in herds from the midwestern United States were seropositive. Young pigs naturally infected by swine HEV were clinically normal but had microscopic evidence of hepatitis, and developed viremia prior to seroconversion. The entire ORFs 2 and 3 were amplified by reverse transcription–PCR from sera of naturally infected pigs. The putative capsid gene (ORF2) of swine HEV shared about 79–80% sequence identity at the nucleotide level and 90–92% identity at the amino acid level with human HEV strains. The small ORF3 of swine HEV had 83–85% nucleotide sequence identity and 77–82% amino acid identity with human HEV strains. Phylogenetic analyses showed that swine HEV is closely related to, but distinct from, human HEV strains. The discovery of swine HEV not only has implications for HEV vaccine development, diagnosis, and biology, but also raises a potential public health concern for zoonosis or xenozoonosis following xenotransplantation with pig organs.

Production of infectious human respiratory syncytial virus from cloned cDNA confirms an essential role for the transcription elongation factor from the 5' proximal open reading frame of the M2 mRNA in gene expression and provides a capability for vaccine development.

Infectious human respiratory syncytial virus (RSV) was produced by the intracellular coexpression of five plasmid-borne cDNAs. One cDNA encoded a complete positive-sense version of the RSV genome (corresponding to the replicative intermediate RNA or antigenome), and each of the other four encoded a separate RSV protein, namely, the major nucleocapsid N protein, the nucleocapsid P phosphoprotein, the major polymerase L protein, or the protein from the 5' proximal open reading frame of the M2 mRNA [M2(ORF1)]. RSV was not produced if any of the five plasmids was omitted. The requirement for the M2(ORF1) protein is consistent with its recent identification as a transcription elongation factor and confirms its importance for RSV gene expression. It should thus be possible to introduce defined changes into infectious RSV. This should be useful for basic studies of RSV molecular biology and pathogenesis; in addition, there are immediate applications to the development of live attenuated vaccine strains bearing predetermined defined attenuating mutations.

Using the International Classification of Functioning, Disability and Health as a tool for analysis of the effect of physical therapy on spasticity in HAM/TSP patients

INTRODUCTION: This study aimed to evaluate spasticity in human T-lymphotropic virus type 1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) patients before and after physical therapy using the International Classification of Functioning, Disability and Health (ICF). METHODS: Nine subjects underwent physical therapy. Spasticity was evaluated using the Modified Ashworth Scale. The obtained scores were converted into ICF body functions scores. RESULTS: The majority of subjects had a high degree of spasticity in the quadriceps muscles. According to the ICF codes, the spasticity decreased after 20 sessions of physical therapy. CONCLUSIONS: The ICF was effective in evaluating spasticity in HAM/TSP patients.

Number of recent sexual partners among blood donors in Brazil: associations with donor demographics, donation characteristics, and infectious disease markers

BACKGROUND: Brazilian blood centers ask candidate blood donors about the number of sexual partners in the past 12 months. Candidates who report a number over the limit are deferred. We studied the implications of this practice on blood safety. STUDY DESIGN AND METHODS: We analyzed demographic characteristics, number of heterosexual partners, and disease marker rates among 689,868 donations from three Brazilian centers between July 2007 and December 2009. Donors were grouped based on maximum number of partners allowed in the past 12 months for each center. Chi-square and logistic regression analysis were conducted to examine associations between demographic characteristics, number of sex partners, and individual and overall positive markers rates for human immunodeficiency virus (HIV), human T-lymphotropic virus Types 1 and 2, hepatitis B virus, hepatitis C virus, and syphilis. RESULTS: First-time, younger, and more educated donors were associated with a higher number of recent sexual partners, as was male sex in Sao Paulo and Recife (p < 0.001). Serologic markers for HIV and syphilis and overall were associated with multiple partners in Sao Paulo and Recife (p < 0.001), but not in Belo Horizonte (p = 0.05, p = 0.94, and p = 0.75, respectively). In logistic regression analysis, number of recent sexual partners was associated with positive serologic markers (adjusted odds ratio [AOR], 1.2-1.5), especially HIV (AOR, 1.9-4.4). CONCLUSIONS: Number of recent heterosexual partners was associated with HIV positivity and overall rates of serologic markers of sexually transmitted infections. The association was not consistent across centers, making it difficult to define the best cutoff value. These findings suggest the use of recent heterosexual contacts as a potentially important deferral criterion to improve blood safety in Brazil.